Repair and regeneration of the injured heart with new, functional cardiomyocytes remains a daunting challenge for cardiovascular medicine. Following cardiac injury, fibroblasts enter injury zone and through various processes actively impair contractile function. Converting cardiac fibroblasts within scar tissue into functional cardiomyocytes is a therapeutic approach that has great potential to restore the function of an injured heart. We were the first to identify a combination of miRNAs, miR combo (miR-1, miR-133a, miR-208a, and miR-499- 5p) that directly reprogrammed cardiac fibroblasts into cardiomyocytes, whereas others have used transcription factors to the same effect. Importantly, delivery of miR combo using lentivirus or retrovirus led to a modest, but significant, improvement in cardiac function. This application is focused on improving miR combo as a therapeutic by identifying ways to enhance efficiency, cell specificity, and effectiveness. Preliminary studies have identified an Adeno-associated virus (AAV) serotype that specifically targets fibroblasts and, moreover, displays enhanced transduction efficiency in vivo. Furthermore, TLR3 activation, which strongly augments miR combo directed reprogramming in vitro, may be a novel way to increase effectiveness. Finally, several transcriptional inhibitors have been identified as miR combo targets. These transcriptional inhibitors repressed the expression of key cardiac transcription factors, suggesting a mechanism for miR combo directed reprogramming. Three overlapping areas will be investigated in this project. Aim 1 will focus on optimizing the efficiency, effectiveness, and cell specificity of miR combo in vitro. To that end we will test if the self- complementary (sc)AAV 2/1 expressing a polycistronic miR combo results in the fibroblast-specific delivery of miR combo. Pharmacological agents will define the appropriate level and duration of TLR3 activation for optimal enhancement of miR combo mediated reprogramming. Aim 2 will address the same questions in vivo. Speckle tracking echocardiography will determine changes in regional wall motion in vivo. Histology will quantify fibrosis levels and lineage tracing will evaluate reprogramming of fibroblasts into cardiomyocytes. Pharmacological agents will be used to determine if TLR3 activation enhances the functional improvements that follow fibroblast reprogramming by miR combo. Aim 3 will define the mechanism by which miR combo promotes reprogramming. Gain- and loss-of-function approaches will determine the role of the transcriptional repressors in miR combo directed reprogramming. Luciferase assays will be used to delineate the physical interaction between the miRNAs within miR combo and the transcriptional repressors. Decoy inhibitor approaches will define the role of NF?B, the key transcription factor in the TLR3 pathway, in miR combo directed reprogramming. Findings from these studies will provide important new insights into improving the efficiency, effectiveness, and cell specificity of direct reprogramming for cardiac repair and regeneration as a therapy.